(1) Field of the Invention
This invention relates generally to the regulation of apoptosis and to compounds which modulate apoptosis, both antagonists and agonists, and more particularly, to a method for identifying compounds with pro-apoptotic or anti-apoptotic activity.
(2) Description of the Related Art
Programmed cell death, referred to as apoptosis, plays an indispensable role in the development and maintenance of homeostasis within all multicellular organisms (Raff, Nature 356:397-400, 1992). Genetic and molecular analysis from nematodes to humans has indicated that the apoptotic pathway of cellular suicide is highly conserved (Hengartner and Horvitz, Cell 76:1107-1114, 1994). In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing the emergence of cancer.
The BCL-2 family of proteins constitutes an intracellular checkpoint of apoptosis. The founding member of this family is the apoptosis-inhibiting protein encoded by the bcl-2 protooncogene which was initially isolated from a follicular lymphoma (Bakhshi et al., Cell 41:889-906, 1985; Tsujimoto et al, Science 229:1390-1393, 1985; Cleary and Sklar, Proc Natl Acad Sci USA 82:7439-7443, 1985). The Bcl-2 protein is a 25 kD, integral membrane protein localized to intracellular membranes including mitochondria. This factor extends survival in many different cell types by inhibiting apoptosis elicited by a variety of death-inducing stimuli (Korsmcyer, Blood 80:879-886, 1992).
The family of BCL-2-related proteins is comprised of both anti-apoptotic and pro-apoptotic members that function in a distal apoptotic pathway common to all multi-cellular organisms. It has been suggested that the ratio of anti-apoptotic (Bcl-2, BCl-x.sub.L, Mcl-1 and A1) to pro-apoptotic (Bax, Bak, Bcl-x.sub.S, Bad, Bik and Bid) molecules may be involved in determining whether a cell will respond to a proximal apoptotic stimulus. (Oltvai et al., Cell 74:609-619, 1992; Farrow, et al., Curr. Opin. Gen. Dev. 6: 45-49, 1996). Because members of this family can form both homodimers and heterodimers, the latter often between anti- and pro-apoptotic polypeptides, the balance of these homodimers and heterodimers could play a role in regulating apoptosis (Oltvai and Korsmeyer, Cell 79:189-192, 1994).
Members of the BCL-2 family have been defined by sequence homology that is largely based upon conserved motifs termed BCL-Homology domains. (Yin et al, Nature 369:321-323, 1994). BCL-Homology domains 1 and 2 (BH1 and BH2) domains have been shown to be important in dimerization and in modulating apoptosis (Yin et al., supra). A third homology region, BH3, has been found in some family members and shown to be important in dimerization as well as promoting apoptosis (Boyd et al., Oncogene 11:1921-1928; Chittenden et al., Embo J 14:5589-5596, 1995). BH4, the most recently identified homology domain, is present near the amino terminal end of some pro-apoptotic family members (Farrow et al., supra).
All known members of the BCL-2 family other than Bad and Bid have a C-terminal membrane-anchoring tail (TM). BCL-2 family members with a TM are intracellular integral membrane proteins most convincingly localized to mitochondria, the endoplasmic reticulum and the nuclear membrane. The intracellular membrane localization of BCL-2 family members together with the identification of structural similarity between the BCl-x.sub.L monomer and the ion-pore forming toxins of colicin and diphtheria toxin B fragment (Muchmore et al., Nature 381:335-341, 1996) has prompted electrophysiological studies by several groups on the ability of BCL-2 family members to form ion channels in artificial lipid membranes.
For example, the anti-apoptotic family members Bcl-x.sub.L and Bcl-2 lacking the TM (Bcl-x.sub.L .DELTA.TM and Bcl-2.DELTA.TM) were shown to insert into synthetic lipid vesicles at pH values below 5.5, but had little or no detectable pore forming activity when added to lipid vesicles at pH values above 5.5 (Minn et al., Nature 385:353-356, 1997; Schendel et al., PNAS USA:94, 5113, 1997; and Antonsson et al., Science 277:370-372, 1997). When added to planar lipid bilayers at physiological pH, Bcl-x.sub.L .DELTA.TM formed channels which exhibit multiple conductance states and which have an ion selectivity sequence of K.sup.+ =Na.sup.+ &gt;Ca.sup.2+ &gt;Cl.sup.- (Minn et al., supra). Bcl-2.DELTA.TM also formed a channel in lipid bilayers at physiological pH having multiple channel conductance states, with a primary conductance of 18.+-.2 pS being consistent with pore formation by Bcl-2 homodimers (Schendel et al., supra). Schendel et al reported that the Bcl-2.DELTA.TM channel is cation selective at pH 5.4 but did not test ion selectivity at neutral pH.
In addition, it has been reported that Bax.DELTA.TM had an intrinsic pore-forming activity in liposomes that was antagonized by Bcl-2 at physiological pH (Antonsson et al., supra). This reference also reported that Bax.DELTA.TM formed voltage-dependent channels in planar lipid bilayers at pH 7.0 that were slightly cation selective, with a permeability ratio of Na.sup.+ to Cl.sup.- of about 2.1.
These electrophysiological studies have led to speculation that Bcl-2 related proteins may modulate apoptosis by regulating the permeability of intracellular membranes in which they are localized. For example, it has been suggested that both Bcl-2 and Bax may allow transport of an ion or a protein across membranes, but in a cytoprotective or a cytodestructive direction, respectively (Schendel et al., supra). In addition, one group speculated that the development of specific Bax channel blockers may be of therapeutic utility in the treatment of neuronal apoptosis (Antonsson et al., supra).
However, until the work reported herein, no direct cause and effect relationship had been shown for the ion conductance state of a channel formed in synthetic planar lipid bilayers by a BCL-2 family member and the modulation of apoptosis. Moreover, there was no description of how to recognize a candidate death agonist or death antagonist by its affect on the ion conductance state of lipid bilayer channels formed by BCL-2 family members.
Some disease conditions are believed to be related to the development of a defective down-regulation of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals. Furthermore, some DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitize the host cellular machinery to drive their own replication and at the same time modulate apoptosis to repress cell death and allow the target cell to reproduce the virus. Moreover, certain disease conditions such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, inflammation, autoimmune diseases and the like may result from a down regulation of cell death regulation. In such disease conditions it would be desirable to promote apoptotic mechanisms.
Conversely, in other disease conditions it would be desirable to inhibit apoptosis such as in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like. In the treatment of such diseases it would be desirable to inhibit apoptotic mechanisms.
Thus, it would be desirable to elucidate the biochemical mechanisms involved in the regulation of apoptosis by BCL-2 family members and to utilize these mechanisms as a basis for identifying compounds which promote or inhibit death. Such compounds would be useful in developing treatment regimens for advantageously modulating the apoptotic process in disease conditions involving either inappropriate repression or inappropriate enhancement of cell death.